1. Field of the Invention
The present invention relates to a novel microorganism which belongs to Aspergillus genus and shows a resistance to both cerulenin and L-methionine analogue and a process for preparing mevinolinic acid therefrom, more specifically, to a novel mutant of Aspergillus terreus resistant to both cerulenin and L-methionine analogue, which provides a high productivity of mevinolinic acid while reducing the production of its analogues, and a process for preparing mevinolinic acid which comprises aerobic culture of the mutant strain and recovery of mevinolinic acid.
2. Description of the Prior Art
Lovastatin(which is also called as mevinolin) is a useful substance for the treatment of human hypercholesterolemia, hyperlipemia, etc., which plays a role as an inhibitor of HMG-CoA(3-hydroxy-3-methylglutaryl-Coenzyme A) reductase, one of enzymes involved in the rate-determining step of biosynthesis of cholesterol in human body. It is prepared by lactonization of mevinolinic acid which is produced by fermentation of molds or microorganisms belonging to Aspergillus terreus(see: Korean patent publication No. 83-2438; U.S. Pat. No. 4,231,938) and Monascus genus (see: Korean patent publication No. 83-2329). Lovastatin is also a starting material for the synthesis of Simvastatin, an inhibitor of HMG-COA reductase(see: Korean patent publication No. 85-669; U.S. Pat. No. 4,444,784).
Lovastatin is chemically represented as 1S-1.alpha.(R*),3.alpha.,7.beta.,8.beta.(2S*,4S*),8.alpha..beta.!!-2-met hylbutanoic acid 1,2,3,7,8,8a-hexahydro-3,7-dimethyl-8-2-(tetrahydro-4-hydroxy-6-oxo-2H-py ran-2-yl)ethyl!-1-naphthalenyl ester, and strains producing the compound also produce an acid form of lovastatin, i.e., mevinolinic acid, which is converted into lovastatin(mevinolin) as a lactone form in the course of isolation and recovery.
Mevinolinic acid produced from Aspergillus terreus is biosynthesized from acetate precursor via polyketide pathway in a similar manner as in the biosynthesis of fatty acid, and it has a chemical structure comprising a backbone derived from 9 acetate units and a side chain consisting of 2 acetate units(see: Moore et al., J. Am. Chem. Soc., 107:3694-3701(1985); Yoshizawa et al., J. Am. Chem. Soc., 116:2693-2694(1994)), and 2 methyl groups transferred from S-adenosyl methionine("SAM")(see: Ming-Shi Shiao and Hsiao-Sheck Pon, Proc. Natl. Sci. Counc. B. ROC, 11(3):223-231(1987)).
A triol polyketide synthase which plays a role in forming a backbone including 1 methyl group, except a side chain(.alpha.-methylbutyrate) in the structure of mevinolinic acid derived from acetate, and its structural gene were isolated from Aspergillus terreus. It was revealed that its amino acid sequence and presumed active site are almost similar to those of a fatty acid synthetase(see: WO95/12661). Also, it was found that methyltransferation occurs from L-methionine through SAM after formation of polyketides consisting of 18 carbons and 4 carbons, respectively, during the biosynthesis of mevinolinic acid(see: Ming-Shi Shiao and Hsiao-Sheck Pon, Proc. Natl. Sci. Counc. B. ROC, 11(3):223-231(1987)).
On the other hand, it has been known that cerulenin blocks biosynthesis of sterol by inhibiting the action of HMG-CoA reductase(see: Ohno et al., Biochemical and Biophysical Research Communications, 57(4):1119-1124(1974)) and inhibits the action of fatty acid synthetase by binding covalently with a cysteine residue of the enzyme which is involved in condensation step during the biosynthesis of fatty acid(see: Funabashi et al., J. Biochem., 105:751-755(1989)).
Moreover, cerulenin, an inhibitor of biosyntheses of fatty acid and sterol also inhibits polyketide biosynthesis(see: Satoshi Omura, Bacteriol. Rev., 40(3):681-697(1976)). Also, it has been found that biosyntheses of leucomycin(see: Omura et al., J. Biochem., 75:193-195(1974)), 6-methylsalicylic acid(see: Ohno et al., J. Biochem., 78:1149-1152(1975)), candicidin(see: Martin et al., Biochimica et Biophysica Acta, 411:186-194(1975)), flavanone(see: Kreuzaler, F & K. Hahlbroch, Eur. J. Biochem., 56:205-213(1975)) and alternariol(see: Hiltunen et al., Applied and Environmental Microbiology, 58(3):1043-1045(1992)), all of which pass through the pathway of polyketide biosynthesis, are inhibited by cerulenin.
Although cerulenin inhibits biosynthesis of fatty acid, Cephalosporium caerulens, a microorganism producing cerulenin can grow even in a medium containing a high concentration(about 100 .mu.g/ml) of cerulenin, and it was reported that such a resistance to cerulenin results from the resistance of its fatty acid synthetase to cerulenin(see: Kawaguchi et al., Archives of Biochemistry and Biophysics, 197(1):30-35(1979)). Also, characterization of a cerulenin-resistant strain isolated from Candida albicans revealed that its resistance to cerulenin results from remarkable decrease of intracellular uptake of cerulenin and decrease of inhibitory action of cerulenin on fatty acid synthetase(see: Cail McElhaney-Feser and Ronald L., Cihlar, Microbiology, 141:1553-1558(1995)).
On the other hand, Aspergillus terreus ATCC 20541 and ATCC 20542 known as microorganisms producing mevinolinic acid, or other known mevinolinic acid producing strains(see: Korean patent publication No. 83-2438; U.S. Pat. No. 4,231,938; Menaghan et al., Can. J. Bot., 73(suppl.1):S925-S931(1995)) have shortcomings of low productivity of mevinolinic acid(for example, productivity of mevinolinic acid of Aspergillus terreus ATCC 20541 is 10-80 .mu.g/ml, and that of Aspergillus terreus ATCC 20542 is 50-850 .mu.g/ml) and high production of mevinolinic acid analogues. In this regard, although it has been reported that a mutant of Aspergillus terreusATCC 20542 obtained by conventional mutation and screening technologies, produced mevenolinic acid in increase of 20% compared with a mother strain, while reducing the production of its analogues such as sulochrin in decrease of 83%(see: Vinci et al., Journal of Industrial Microbiology, 8:113-120(1991)), the mutant strain has proved to be less satisfactory in a sense that it still produces some analogues including triol acid highly, while providing a low productivity of mevinolinic acid. Accordingly, there are strong reasons for exploring and developing alternative means for high production of mevinolinic acid, while reducing the production of its analogues such as asterric acid, butyrolactone, citrinin, emodin, itaconic acid, geodin, sulochrin, terretonin, etc.